| Homologous recombination (HR) involves the exchange of genetic information between two similar or identical DNA sequences, and is important in the maintenance of genome stability. Homologous recombination may occur by gene conversion, a non-reciprocal form of recombination in which genetic information is transferred from a donor to a recipient sequence, or by crossing over, a mechanism that generates reciprocal recombination products. In this thesis, a well-defined murine hybridoma cell culture system was exploited to test two hypotheses related to HR, (1) spontaneous gene conversion involves faithful genetic information transfer, and (2) the eukaryotic strand exchange protein, Rad51 is important in the HR process.; Intrachromosomal gene conversion between genetically-marked heteroallelic sequences occurs with high fidelity and results in long gene conversion tracts, which have continuous transfer of genetic information in favour of the donor sequence. In some recombinants, evidence of intrachromatid gene conversion was obtained. In these respects, spontaneous mitotic gene conversion in mammalian cells closely resembles equivalent gene conversion events in the yeast, Saccharomyces cerevisiae, but contrasts with double-strand break (DSB)-induced gene conversion in yeast and mammalian cells. The studies reported here support faithful transfer of genetic information between donor and recipient sequences during spontaneous intrachromosomal gene conversion in mammalian cells.; To test the requirement for Rad51 in HR, mouse hybridoma cells stably expressing wildtype Rad51, or the Walker box lysine variants, Rad51-K133A and Rad51-K133R, deficient in ATP binding and ATP hydrolysis, respectively, were constructed. A unique feature is the recovery of transformants expressing Rad51-K133A. Augmentation of the endogenous pool of Rad51 by expression of transgene-encoded wildtype Rad51 enhances cell growth and gene targeting, but has minimal effects on cell survival to DNA damage induced by ionizing radiation (IR) or mitomycin C (MMC). Whereas expression of Rad51-K133A impedes growth, in general neither Rad51-K133A nor Rad51-K133R significantly affected survival to IR- or MMC-induced damage, but did significantly reduce gene targeting. Expression of wildtype Rad51, Rad51-K133A or Rad51-KI33R did not affect the frequency of intrachromosomal HR. However, in both gene targeting and intrachromosomal HR, wildtype and mutant Rad51 transgene expression altered the recombination mechanism: in gene targeting, wildtype Rad51 expression stimulates crossing over, while expression of Rad51-K133A or Rad51-K133R perturbs gene conversion; in intrachromosomal HR, cell lines expressing wildtype Rad51, Rad51-K133A or Rad51-K133R display increased deletion formation by intrachromosomal HR. The results suggest that ATP hydrolysis by Rad51 is more important for some HR functions than it is for other aspects of DNA repair. |
